The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
In vitro fertilization is a technique used to overcome various forms of male and female infertility. The process involves fertilizing an oocyte with a sperm in vitro and subsequently transferring the developing embryo into the female body. Even though it has been nearly 30 years since the first birth from IVF (Steptoe, P. C. and Edwards, R. G., Lancet 2(8085): 366 (1978)), the process faces continuing challenges of low implantation and pregnancy rates. In the first ten years following the birth of the first “test tube baby,” the reported success rate for IVF was only 8 to 10%. This low success rate was due, at least in part, to the poor quality of the embryo culture media. Unfortunately, despite a continuous effort to improve and specialize IVF culture media over the last 30 years, the success rate has only increased to approximately 30 to 35% in instances where single transfer is employed (based on national IVF registers in Finland and Sweden 2005). This single transfer data is the data most comparable to the 1978 data. This slow progress in improving IVF success rates reflects the difficulty and unpredictability in altering and enhancing culture media for developmental cells, such as gametes, zygotes and embryos.
The types of media presently used for human IVF have fallen into two categories; simple and complex. Simple media are those, such as Earle's and human tubal fluid (HTF), which are balanced salt solutions with added carbohydrate energy sources such as pyruvate, lactate and glucose. Complex media, such as Ham's F-10, further include non-essential and essential amino acids as well as other additives, such as vitamins, antibiotics and serum or proteins.
Although some IVF culture media are intended to support embryo development up to the 8-cell stage or beyond in a single medium, the trend has been to optimize separate culture media to support the developing embryo at different stages of development. This has led to the widespread use of sequential culture medium in IVF. For example, a sequential culture media system may use one culture medium for the growth of an embryo from a one-cell zygote to an eight-cell embryo during the first 48 hours of development and another culture medium to grow the eight-cell embryo to the blastocyst stage. As the female reproductive tract provides a changing environment for the developing embryo, the sequential culture media are designed to more closely mimic the female reproductive tract during in vivo embryo growth. The media compositions typically differ with respect to components such as amino acids and sugars to improve optimization of the media to support the embryonic growth and development.
The culture media employed in IVF have undergone some evolution over the course of the last 30 years. However, in some respects the culture media have remained strikingly unchanged. The focus of most of the research aimed at improving embryo culture media has been on the components that make up the core or bulk of the media—carbohydrate energy sources, amino acids, serum and salts/buffers. Other ingredients have received far less attention. In some cases it appears that these ingredients have been included in embryo culture media simply because they were present in the early somatic cell culture media from which embryo culture media initially evolved. Lipoic acid (LA) is one such ingredient.
Lipoic acid, also known as α-lipoic acid or thioctic acid, is widely known for its role as the coenzyme of the E2 (dihydrolipoate acyltransferase) subunit of multienzymatic complexes catalyzing oxidative decarboxylation of pyruvate, α-ketoglutarate, and branched-chain α-ketoacids. However, over the past ten years or so, it has become evident that lipoic acid is also an antioxidant. The efficiency of lipoic acid has been attributed to unique antioxidant properties of the lipoate/dihydrolipoate system, its reactive oxygen species (ROS) scavenging ability, and significant effect on the tissue concentrations of reduced forms of other antioxidants, including one of the most powerful, glutathione. Data from the literature suggests that lipoic acid and dihydrolipoate (DHLA) may or may not have growth promoting effects on specific cell types as murine leukemic cells and Jurkat T cells. Steptoe, P. C. and Edwards, R. G., Lancet 2(8085): 366 (1978).
Lipoic acid is a disulfide, amphiphilic compound which contains a chiral center creating two enantiomeric forms (R and L), with the most biologically active being the R-enantiomer. The disulfide component provides the molecule with metal-chelating properties while the compound's amphiphilic properties allow the relatively small molecule (MW=206.34 g/mol) to readily diffuse across the cell membrane where it is converted easily to its dithiol, reduced form, DHLA by several cellular multienzyme complexes involved in the decarboxylation of α-ketoacids; aerobically important steps in energy metabolism. Additionally, the DHLA/LA couple has a reported redox potential of −0.29 V making the pair a strong electron acceptor/oxygen radical scavenger unit. This strong redox potential allows the molecule to act as a potential recycler of other antioxidants such as vitamin C, vitamin E, glutathione, coenzyme Q10, and ubiquinone. Although the reduction of lipoic acid is presumed to take place within cells, the DHLA generated is thought to leak from the cells into the surrounding medium implicating both intracellular and extracellular antioxidant capabilities.
Lipoic acid is universally present in prokaryotes and eukaryotes. Lipoate, the unprotonated base, is the most prevalent form at physiological conditions and is thought to be synthesized within the cell by lipoic acid synthetase from the precursors' octanoic acid and a sulfur source, most likely a cysteine residue. This makes the molecule a non-essential nutrient, although it has been reported to be a valuable dietary supplement in diseases associated with excessive oxidative stress, including arthritis, diabetes, atherosclerosis, metabolic syndrome, and Alzheimer's. See, e.g., Pershadsingh H A, Expert Opin Investig Drugs 16:291-302 (2007); Holquist et al., Pharmacol Ther. 113: 154-64 (2007).
Despite the increasing body of knowledge regarding the role of lipoic acid in the body, very little is known about its effect on the growth of developmental cells, such as embryos, in culture. Although lipoic acid is sometimes included at low concentrations in embryo culture media, it is usually absent. The two studies that have systematically investigated the role of lipoic acid in embryo culture media concluded that the omission of lipoic acid from an embryo culture medium did not have an effect on outcome. The first study looked at the effect of 11 water-soluble vitamins, including lipoic acid, from Ham's F10 medium on the development of 8-cell hamster embryos to hatching and hatched blastocysts in vitro. (Kane et al., Biology of Reproduction, 39, 1137 (1988).) The study concluded that the omission of lipoic acid on the development of 8-cell hamster embryos resulted in no significant effect on the development at any stage of culture. The second study observed the effect of the omission of the 11 water-soluble vitamins, including lipoic acid, of Ham's F10 medium on the culture of rabbit morulae to expanded blastocysts. (Kane, J. of Expt. Zoology, 245, 220 (1988).) This study also concluded that there was no significant effect due to the omission of lipoic acid. Nonetheless, lipoic acid has been included in some embryo culture media.
For reasons that are not clear from the published literature, those embryo culture media that include lipoic acid limit the concentration to 1 μM or lower. This is likely due to the fact that many embryo culture media initially evolved from Ham's F10 and F12 media, early and still common somatic culture media, that happened to include lipoic acid at a concentration of 1 μM. However, it is clear from the studies cited above that the necessity or even desirability of including lipoic acid in such media was not a consideration during the early adoption of Ham's F10 and F12 media as the base for early embryo culture media. This is also clearly shown by the fact that none of the current major commercial embryo culture media products appear to contain any lipoic acid at all. In fact, the trend in the evolution of somatic cell culture media from Ham's F10 and F12 has also been toward dilution of those media, resulting in culture media with even lower concentrations of lipoic acid. For example, a popular culture medium that followed Ham's F12 is DMEM/F12 which is a 1+1 dilution of Ham's 12 with Dulbecco's modified Minimal Essential Medium (DMEM), another culture medium that is free of lipoic acid. RDF is another common culture medium that came later and built on DMEM/F12. RDF is a 1+1 dilution of DMEM/F12 with RPMI 1640, another culture medium that is free of lipoic acid. Thus, trends in the evolution of culture media from Ham's F10 and F12 demonstrate that the role of lipoic acid in culture media has been overlooked and unappreciated.
Lipoic acid has also been included in somatic cell culture media at other concentrations, however, the concentration of lipoic acid used in such media varies over many orders of magnitude and it has been acknowledged that “successful in vitro culture of different cell types will often require the use of different media formulations.” (See, for example, PCT application publication number WO 98/08934.) This is particularly true for culture media for developmental cells, such as embryos, which have very different requirements from somatic cells. In addition, even within the somatic cell culture field, there is disagreement as to whether lipoic acid is beneficial in a culture medium. (See, for example, PCT application publication number WO 99/35242, page 44, stating “α-Lipoic acid (thioctic acid) is sometimes added to culture media, but there is little evidence that it is actually needed.”) As such, the literature regarding the use of lipoic acid in somatic cell culture media sheds no light on the desirability or effect of lipoic acid in culture media for developmental cells, such as gametes, zygotes and embryos.